Voltage-gated sodium or Nav channels are fundamental components of signaling in mammalian central neurons in that they confer electrical excitability to neurons. Their activity changes in epileptic neurons, and they are prominent targets of anti-epileptic drugs. This proposal is aimed at determining the fundamental mechanisms that regulate the function of the Nav1.2 channel, which plays a crucial role in the excitability of axons and nerve terminals in many mammalian central neurons. Using novel, state-of-the-art mass spectrometric approaches we have discovered an unanticipated complexity of in vivo phosphorylation on Nav1.2 purified from rat brain. These data provide the first opportunity to investigate the role of these novel and unambiguously identified in vivo brain phosphosites on Nav1.2 in governing channel gating. Of particular interest is determining whether these phosphorylation sites regulate the switch between the transient Nav channels that predominate in normal mammalian neurons, and the persistent Nav channels more typical of epileptic neurons. We will also for the first time undertake quantitative proteomic analyses of how in vivo phosphorylation changes in animal models of acute epileptic seizures, and of epileptogenesis in the form of spontaneous recurrent seizures that serve as animal models of human temporal lobe epilepsy. These studies will yield important insights into the physiological and pathological regulation of Nav1.2 channels, which are key regulators of neuronal excitability in mammalian brain and important targets for anti-epileptic drugs. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for epilepsy.